The long-term goal of this project is to understand the mechanisms underlying CD45 alternative splicing and the role of CD45 splicing in lymphocyte and haematopoietic cell biology. CD45, a transmembrane tyrosine phosphatase that is broadly expressed in haematopoietic cells, is an essential modulator of signal transduction through the T and B cell antigen receptors (TCR, BCR). The CD45 gene consists of ~33 exons, of which exons 4, 5 and 6, encoding regions A, B and C of the extracellular domain respectively, can be variably spliced out. Naive primary T cells express the larger isoforms (RABC, AB, BC, etc) whereas activated/ memory T cells and most T cell lines express the shortest isoform (RO). In contrast immature and mature B cells and most B cell lines express only the largest isoform, CD45RABC (commonly known as B220). CD45 phosphatase activity is required to initiate as well as modulate TCR and BCR signalling, and this function depends on the particular splice variants of CD45 expressed. In this application we build on our recent discovery that the heterogenous nuclear ribonucleoprotein HNRPLL is the primary regulator of CD45 alternative splicing in stimulated T cells. Using a Jurkat cell line in which expression of CD45 isoforms is sensitive to PMA stimulation, we performed a limited RNAi screen which identified HNRPLL as a major regulator of CD45 alternative splicing. We have shown that HNRPLL expression is tightly associated with exclusion (skipping) of exons 4-6. HNRPLL expression is upregulated by PMA stimulation, and in turn, HNRPLL upregulates CD45RO and downregulates CD45RABC and other isoforms which retain the intermediate exons 4-6. HNRPLL knockdown has the opposite effect, decreasing CD45RO expression and maintaining the expression of CD45RABC and other isoforms containing exons 4-6. We will follow up on this discovery in the proposed project. Specifically, in Aim 1 we will define the molecular mechanism of action of HNRPLL, identifying the components of the inducible splicing complex containing HNRPLL and defining its pre-RNA targets; in Aim 2 we will perform additional RNAi screens to identify the signalling pathways and transcription factor(s) that modulate CD45 splicing; in Aim 3 we will investigate the role of HNRPLL in T and B cells in vivo by generating mice conditionally expressing HNRPLL from the ROSA26 locus or bearing a conditional allele of HNRPLL for deletion in different tissues; and in Aim 4 we will ask whether changes in HNRPLL expression modulate T and B cell signalling. Together these findings have the potential to increase our general understanding of how signalling pathways regulate mRNA splicing, as well our specific knowledge of the role of CD45 splice isoforms in haematopoietic cell biology. PUBLIC HEALTH RELEVANCE: In most genes in higher organisms, the pieces of DNA that code for proteins (known as exons) are separated by intervening regions known as introns, which need to be removed through a process called gene splicing so that the exons can be joined (spliced) together and the encoded protein can be made. In this proposal we plan to examine a particular protein, known as HNRPLL, that we have shown is involved in gene splicing in T and B cells of the immune system. We will investigate the various properties of HNRPLL -- such as RNA-binding and association with other proteins -- that allow it to perform its particular functions; define the signalling mechanisms that allow it to be turned on when T cells encounter antigens; and study the importance of HNRPLL for the health of the organism by generating mice lacking or expressing HNRPLL in particular organs and tissues.